SEARCH · Search PubMed

Results for “Spin Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources

PubMed1981

Spatial separation of the two essential thiol groups and the binding site of the exchangeable GTP in brain tubulin. A spin label study.

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu2+ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards N-ethylmaleimide and p-chloromercuribenzoate, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by N-ethylmaleimide, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg2+, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of N-ethylmaleimide. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn2+ for Mg2+. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups. b. The binding sites of the exchangeable GTP and Mg2+ are located 10 A, at least, from the two most reactive sulfhydryl groups. c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 A. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.

Animals↗

PubMed1988

Pharmacokinetics of a spin-labeled rubomycin analog.

Pharmacokinetics of a spin-labeled analog of rubomycin (ruboxyl) was studied. Differences were found in ruboxyl pharmacokinetics in normal and tumor-bearing animals. Most of the drug was excreted within 6 h. The differences in pharmacokinetics of ruboxyl and nitroxyl radical were established.

Animals↗

PubMed1999

Effects of conjugated linoleic acid on oxygen diffusion-concentration product and depletion in membranes by using electron spin resonance spin-label oximetry.

The effect of conjugated linoleic acid (CLA) on the relation between structure and function of membranes is described in this paper. Electron spin resonance (ESR) spin-label oximetry was used in the present study to evaluate if oxygen transport and oxygen depletion were affected by incorporation of CLA instead of linoleic acid into membrane phospholipids. Specifically, 1-stearoyl-2-(9cis, 11 trans-octadecadienoyl)-phosphorylcholine (SCLAPC) was incorporated into soy plant phosphatidylcholine (soy PC) or egg yolk PC (EYPC) bilayers. The use of spin labels attached to different carbons along the fatty acid chain makes it possible to carry out structural and oximetric determinations with the same test sample. For example, the incorporation of 5 mol% SCLAPC increased the oxygen diffusion-concentration product in soy PC or EYPC liposomes at 37 degrees C, slightly decreased the ordering of the hydrocarbon chains at the C10 and C12 positions (in the region of the conjugated double bonds), and increased the rate of oxygen depletion from the aqueous medium. Similar results were not obtained by incorporating 5 mol% of 1-stearoyl-2-linoleoyl-PC (SLPC). In our model system, free-radical generation was initiated by extended incubation of the liposomes, by induction by 2,2'-azobis(2-amidinopropane)hydrochloride, or by ultraviolet irradiation of H2O2. The rate of consumption of molecular oxygen was studied by monitoring the oxygen concentration in the aqueous phases of the liposomes. The effect of 5 mol% SCLAPC in soy PC was significantly larger than 5 mol% SLPC in soy PC; the response patterns with soy PC and EYPC were similar. Furthermore, 5 mol% SCLAPC in 1-palmitoyl-2-linoleoyl-PC showed similar oxygen consumption to that observed with 5 mol% SCLAPC in EYPC. On the other hand, 5 mol% SCLAPC in synthetic PC membranes containing saturated or monounsaturated fatty acids showed low oxygen depletion rates. The perturbation of membrane structure and the increase of the relative oxygen diffusion-concentration products provided a potential mechanism by which CLA incorporated into membrane lipids could affect oxidative stress.

Electron Spin Resonance Spectroscopy↗

PubMed1976

Conformational changes in glycogen phosphorylase studied with a spin-label probe.

Phosphorylase b and a were covalently modified on essentially one -- SH group per subunit by a spin label 4-(2-iodoacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The electron spin resonance spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, beta-glycerol-2-phosphate). The interactions are analysed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.

Adenosine Diphosphate↗

PubMed1979

Protein-cationic detergent interaction. Interaction of bovine serum albumin and other proteins with alkylpyridinium bromides studied by viscosity, gel filtration and spin-label methods.

Viscosity, gel filtration and spin-labelling methods have been used to study the influence of alkylpyridinium bromides on the conformation of bovine serum albumin and other proteins. Cationic detergents cause partial unfolding of the native protein molecules. The magnitude of these changes increases with increasing length of the detergent hydrocarbon chain. When cationic detergents are added to reduced and carboxymethylated bovine serum albumin the observed changes are opposite to those found in native protein.

Animals↗

PubMed1985

Structural changes of rat liver microsomal membranes induced by the oral administration of carbon tetrachloride. 31P-NMR and spin-label studies.

The acute effects of carbon tetrachloride (CCl4) on the membrane structure of rat liver microsomes were studied using 31P-NMR and spin-labeling techniques. 31P-NMR spectra of rat liver microsomes were not changed appreciably after the oral administration of CCl4, indicating that the surface structures of microsomal membranes probably are not influenced by the oral administration of CCl4. Four different spin-labeled stearic acids, 5-(N-oxyl-4',4'-dimethyloxazolidine)-stearic acid (5SLS), its methyl ester (5SLSM), 12-(N-oxyl-4',4'-dimethyloxazolidine)-stearic acid (12SLS) and its methyl ester (12SLSM), were used for the estimation of membrane fluidity. The apparent rotational correlation time of 12SLS decreased from 4.0 nsec to 3.0 nsec after the oral administration of CCl4, while the order parameter of 5SLS did not change. The results suggest that CCl4 or its metabolites increase the membrane fluidity of liver microsomes primarily at hydrophobic regions rather than at the surface layer. The ESR spectrum of 5SLSM in microsomal membranes comprised two different signals; one was an anisotropic signal and the other was a rather isotropic one. The ratio of the anisotropic signal to the isotropic one decreased markedly after the oral administration of CCl4 and depended on the dose of CCl4. The suitability of this ESR technique with 5SLSM for the estimation of membrane damage is discussed.

Administration, Oral↗

PubMed1984

Interactions and spatial arrangement of spin-labeled NAD+ bound to glyceraldehyde-3-phosphate dehydrogenase. Comparison of EPR and X-ray modeling data.

The spatial arrangement of coenzyme NAD+ in remote and adjacent binding sites in various stoichiometric complexes with tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was examined via EPR spectroscopy. An adenosine N6-15N,2H17 spin-labeled derivative of coenzyme NAD+ (SL-NAD+) was chemically synthesized for this work. The spectral simplifications and narrow line widths afforded by 15N and 2H substitution enabled experimental EPR spectra to be deconvoluted into their three component spectra: (a) unbound coenzyme, (b) bound coenzyme without adjacent site occupied, and (c) bound coenzyme with adjacent site occupied. Binding of SL-NAD+ in adjacent active centers of R axis-related subunits resulted in resolved dipolar interactions which characterized intersubunit distances. Binding to distant subunits related by the P and Q axes gave no dipolar interaction. Once the first NAD+ site was occupied, EPR spectra at various stoichiometries provided evidence for nonpreferential spatial binding of SL-NAD+ to the three unoccupied sites. EPR spectral simulations indicated a separation of 12.8 A for the unpaired electrons of spin label moieties of R axis-related coenzymes. Molecular modeling based on x-ray crystallographic data predicted 11-13 A. The angles and distance relating to interacting spin-labels were calculated from atomic coordinates based on molecular modeling of both anti-anti and anti-syn (adenine-ribose) conformations of SL-NAD+. Computer-generated line shapes indicated best agreement with experimental EPR results when the anti-anti geometry was employed. Comparison of EPR spectra from soluble and ammonium sulfate-precipitated enzymes indicated that the NAD+-binding domains are positioned equivalently in the two physical states. Since the observed dipolar line shapes are critically dependent on the distance and geometry relating to the interacting SL-NAD+, these data provide direct evidence for a high degree of conservation of quaternary structure of the enzyme in the hydrated crystalline state. Studies on the enzyme isolated from human erythrocytes also indicated a close correlation with the rabbit muscle enzyme in both the arrangement of NAD+-binding domains and negative cooperativity of coenzyme binding.

Animals↗

PubMed1989

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines.

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

Bacterial Proteins↗

PubMed1984

Interaction of hexachlorophene and other compounds with spin-labeled brain membranes.

Experiments reported here demonstrate that hexachlorophene influences oxidation-reduction events inside the brain membrane, possibly via a free radical mechanism. This was shown by nitroxide spin label quenching inside the rat cerebellum membrane bilayer due to the interaction between hexachlorophene and peroxidase-hydrogen peroxide system. Prior addition of antioxidants, e.g., vitamin E or butylated hydroxytoluene, prevented such membrane-bound fatty acid spin label reduction, presumably due to their free radical scavenging abilities. The 5-doxyl stearic acid spin probe attached to the brain membranes did not exhibit any detectable changes in their ESR spectra nor, consequently, in the microviscosity of the membranes when exposed to up to 40 mM hexachlorophene.

Animals↗

PubMed1984

Nonaromatic amino acids in the combining site region of a monoclonal anti-spin-label antibody.

The nuclear magnetic resonance spectra of monoclonal Fab antibody fragments have been recorded in the absence and presence of the specific spin-label dinitrophenyl hapten. The difference spectra reveal the presence of about 50 amino acids in the region of the combining site. By selective deuteration and by use of double difference spectra, all the resonances in the spectral region -1 to 1.5 ppm have been identified. We have found that in the combining site region there are four or five valines, certainly three and possibly five threonines, three or four leucines, two or three isoleucines, and six or seven alanines. Selective deuteration of methionine and lysine reveals one methionine and two lysines in the difference spectra. All of these amino acids are estimated to be within 17 A of the paramagnetic hapten. By using difference spectra involving low fractional occupancy of the combining site with the spin-label hapten, it is established that one threonine and one valine are very close to the paramagnetic hapten.

Amino Acids↗

PubMed2005

Explicit treatment of spin labels in modeling of distance constraints from dipolar EPR and DEER.

Current SDSL-EPR methods allow measurement of dipolar distances in the 8-70 A range; however, the use of extrinsic probes complicates the interpretation of these distances in modeling macromolecular structure and conformational changes. The data presented here show that interprobe distances correlate only weakly with Cbeta-Cbeta distances, especially for distances that are on the order of the spin label tether lengths. Explicitly incorporating the spin label into the modeling process increases the experiment/model correlation 4-fold and reduces the distance error from 6 A to 3 A.

Carbon↗

PubMed1982

Orientation of platelets on the surfaces of glass and Teflon plates as studied by a spin label technique.

Platelets were found by a spin label technique to orient on flat surfaces of glass as well as of Teflon. This kind of platelet orientation was not caused by centrifugation or partial dehydration of the membrane preparation as employed usually to make oriented planar multilayers of biological membranes on the surface of a supporting plate (1-8), but was considered to be closely related to the adhesion or the aggregation properties of platelets. The amount of oriented platelets varied depending on the platelet treatment and was estimated from a computer simulation of the observed ESR spectra to be about one-half of that of the non-oriented ones in the case of thrombin-treated platelets. This technique may be useful as a new tool to explore the adhesion or aggregation properties of platelets.

Animals↗

PubMed1999

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach.

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 degrees C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2 p pi orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed.

Biophysical Phenomena↗

PubMed2004

Chromophoric spin-labeled beta-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C beta-lactamases.

The chromophoric spin-label substrate 6-N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M(-1) cm(-1) at 275 nm compared to 670 M(-1) cm(-1) at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/KM, determined under initial velocity conditions, were 637 +/- 36 s(-1) and 13.8 +/- 1.4 x 10(6) M(-1) s(-1), respectively, for hydrolysis catalyzed by TEM-1 beta-lactamase of E. coli, and 0.5 +/- 0.04 s(-1) and 3.9 +/- 0.4 x 10(4) M(-1) s(-1) for hydrolysis catalyzed by the beta-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 beta-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H2O (30:70, v:v) cryosolvent mixtures buffered to pH* 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 degrees C was > or = 3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 beta-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.

Acylation↗

PubMed1969

Magnetic resonance studies of spin-labeled creatine kinase system and interaction of two paramagnetic probes.

Creatine kinase from rabbit muscle has been specifically labeled with a derivative of a nitroxide free radical on the two essential sulfhydryl groups, one per active site. The conformation in the environment of the enzyme-bound free radical and the change in conformation induced upon binding of the substrate, metal adenosine diphosphate, has been monitored by two parameters of the bound spin label, its electron paramagnetic resonance spectrum and its contribution to the proton relaxation rate of water. The specificity in activation of the enzymic reaction of the series of alkaline earth ions, Mg, Ca, Sr, Ba, was not reflected by differences in conformation of the ternary enzyme metal substrate complexes observable with the spin probe. However, with the inhibitory zinc ion, the conformation of ternary complex was distinctly different. The electron paramagnetic resonance spectra of complexes with two paramagnetic probes, namely, spin-labeled enzyme with paramagnetic metal adenosine diphosphate complexes (Mn(II), Ni(II), and Co(II)) revealed a spin-spin interaction between the protein-bound free radical and the paramagnetic metal ions. From the magnitude of the interaction, the distance between the manganous ion and the electron spin of the nitroxide moiety in the MnADP-spin-labeled creatine kinase was estimated to be in the range of 7-10 A.

Adenine Nucleotides↗

PubMed1978

[Studies of spin-labeled spectrin].

Spectrin isolated from human erythrocytes has been spin-labeled with five maleimide nitroxides. The mobility of the labels is strongly dependent on their size, and on the temperature. A thermal transition of spectrin is shown to occur above 30 degrees C. Calcium and magnesium provoke a strong immobilization of the labels. This effect is yet more pronounced when spectrin is allowed to reassociate with the cytoplasmic surface of the membrane.

Electron Spin Resonance Spectroscopy↗

PubMed1994

Reorganization of lipid domain structure in membranes by a transmembrane peptide: an ESR spin label study on the effect of the Escherichia coli outer membrane protein A signal peptide on the fluid lipid domain connectivity in binary mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine.

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their point of connectivity (Sankaram, M.B., D. Marsh, and T.E. Thompson. 1992. Biophys. J. 63:340-349). In the absence of the peptide, the number of fluid lipid domains does not vary with the fraction of lipid that is in the fluid phase, and phase conversion is accomplished solely by changes in the domain size. The phase boundaries of the lipid mixture remain largely unchanged by the presence of the peptide at mole fractions up to 0.02, but both the size and number of the fluid domains is changed, and the point at which they become connected is shifted to lower fractions of the fluid phase. In addition, the number of domains in the presence of the peptide no longer remains constant but increases from a domain density at low fractions of the fluid phase that is much lower than that in the absence of peptide to one that is comparable to the natural state in the absence of peptide at the point of domain connectivity. A simple model is presented for the process of domain fission, where the latter is determined by a balance between the effects of peptide concentration in the fluid domains, the line tension at the domain boundaries, and the distributional entropy of the domains.

Amino Acid Sequence↗

PubMedDate not supplied

[Models for rotating spin labels and probes in proteins and membranes].

A method of comparative analysis of ESR spectra has been proposed. It allows to distinguish between two models, slow reorientation of spin labels, and rapid rotation of the cone. Comparison of experimental data for a number of biological objects the theoretical predictions has shown that the rotation of nitroxyl fragment of spin labels can be described by the model of slow anisotropic rotation with correlation time 10(-6) less than or equal to tau less than or equal to 10(-8) s in conditions where the rotation of macromolecules is "frozen".

Cell Membrane↗